The enterovirus comprises a large genus of viruses which produce a wide range
of diseases. They are part of the larger family of Picornaviridae which also
includes aphtoviruses, cardioviruses, and rhinoviruses. Classically, enterovirus
includes the following diseases as outlined below. Transmission is usually
by ingestion of fecally contaminated material. Incubation periods have ranged
from 2 days to 2 weeks with the majority of infections occurring with 3-5
days following exposure. All of the viruses may cause aysmptomatic infection,
a febrile illness with or without respiratory symptoms, aseptic meningitis,
encephalitis, and paralysis. More specific illnesses have been associated
with some groups or serotypes

A serologic survey was conducted on a population-based representative
sample of 521 18-year-old soldiers recruited to the Israel Defence Forces
in 1997. The prevalence of neutralizing antibodies and geometric mean
titers (GMTs) against the three types of poliovirus (Mahoney, MEF and
Saukett strains) were found to be 98.7% (GMT--169.95), 99.6% (GMT--297.14)
and 96.4% (GMT - 59.48), respectively. These GMTs are markedly lower
than those recorded 4 years after booster vaccination carried out during
a 1988 polio outbreak, and suggest a decline in immunity against polio
among young adults.

These findings support the policy of routine revaccination of children
and adolescents in countries at risk of imported polioviruses and of
revaccination of adults traveling to areas to which polio is endemic.

DISEASE ASSOCIATIONS

CHARACTERIZATION

CARDIAC

Clinical and prognostic significance of detection
of enteroviral RNA in the myocardium of patients with myocarditis or
dilated cardiomyopathy.

BACKGROUND: Enteroviral RNA sequences have been demonstrated in the
myocardium of patients with myocarditis or dilated cardiomyopathy from
presentation to end-stage disease. The prognosis of heart muscle disease
has not previously been evaluated in relation to the detection of enterovirus
in myocardial biopsy tissue.

METHODS AND RESULTS: We studied 123 consecutive patients with heart
muscle disease prospectively. Multiple endomyocardial biopsy samples
taken from all patients during diagnostic cardiac catheterization were
classified histologically and were examined for enteroviral RNA by use
of an enterovirus group-specific hybridization probe. Three enterovirus-negative
patients with cardiac amyloidosis were excluded from subsequent analysis.
Enteroviral RNA sequences were detectable in 41 (34%) of the remaining
120 patients (group A), while 79 (66%) had no virus detected (group
B). The groups did not differ significantly in age, sex, symptomatic
presentation, or hemodynamic characteristics; duration of symptoms was
significantly shorter in group A (7.8 +/- 9.6 versus 14.9 +/- 19.0 months,
P < .05). At follow-up (mean, 25 months; range, 11 to 50 months), patients
from group A had an increased mortality compared with those in group
B (25% versus 4%, respectively; P = .02). Mortality was also statistically
greater in patients with symptomatic cardiac failure (P = .02), those
with elevated left ventricular end-diastolic pressures (P = .03), and
those in New York Heart Association functional classes III and IV (P
= .05). Multivariate regression analysis, however, showed that only
the presence of enterovirus RNA and symptomatic heart failure were of
independent prognostic value.

CONCLUSIONS: These data demonstrate that the detection of enterovirus
RNA in the myocardium of patients with heart muscle disease at the time
of initial investigation is associated with an adverse prognosis and
that the presence of enterovirus RNA is an independent predictor of
clinical outcome.

The Enterovirus may be the most common agent responsible for viral
myocarditis and cardiomyopathy. Very little of the literature is available
concerning the follow-up of patients who underwent transplantation with
enteroviral positivity in native hearts.

In the present study, 45 explanted hearts from patients who underwent
orthotopic heart transplant at University of Padova were studied by
reverse transcriptase (RT)-polymerase chain reaction (PCR): 27 patients
had dilated cardiomyopathy (DC), 12 had ischemic cardiopathy (IC), 2
had valvular disease (VD), 2 had arrhythmogenic right ventricular cardiomyopathy
(ARVC), 1 had giant cell myocarditis (GCM), and 1 had lymphocytic myocarditis
(LM). Two sets of PCR primers from the highly conserved region of Enterovirus
and Rhinovirus were used. Samples of both ventricles and septum were
analyzed in every patients. The RT-PCR and nucleotide sequencing of
amplicons were also performed on all post-transplantation follow-up
biopsies in patients with Enterovirus positivity in the native heart.
The viral genome was detectable in only 1 of 27 patients with DC (3%)
and in 1 patient with LM. Nucleotide sequence analysis of the amplified
product showed differences in nucleotide sequence of PCR samples compared
with the sequence of the coxsackievirus B3 used in the current study.
The patient with Enterovirus-positive DC showed a higher index of severe
rejection (>3A) in the first 6 months, compared with the other patients
tested. The patient with Enterovirus-positive LM died of disease recurrence
2 months after transplantation.

The present study reveals a scarce presence of Enterovirus in the myocardium
of patients with chronic myocardial disease. Because Enterovirus infection
was predictive of a poor prognosis in these two patients, molecular
studies are useful in excluding viral involvement in native hearts of
transplanted patients.

Human coxsackie-adenovirus receptor is colocalized
with integrins alpha(v)beta(3) and alpha(v)beta(5) on the cardiomyocyte
sarcolemma and upregulated in dilated cardiomyopathy: implications for
cardiotropic viral infections.

BACKGROUND: The coxsackievirus and adenovirus receptor (CAR) was identified
as a common cellular receptor for both viruses, but its biological and
pathogenic relevance is uncertain. Knowledge of CAR localization in
the human cardiovascular system is limited but important with respect
to CAR-dependent viral infections and gene transfer using CAR-dependent
viral vectors.

METHODS AND RESULTS: Explanted failing hearts from 13 patients (8 with
dilated cardiomyopathy [DCM] and 5 with other heart diseases [non-DCM])
and normal donor hearts (n=7) were investigated for the expression levels
and subcellular localization of CAR and the adenovirus coreceptors alpha(v)beta(3)
and alpha(v)beta(5) integrins. CAR immunoreactivity was very low in
normal and non-DCM hearts, whereas strong CAR signals occurred at the
intercalated discs and sarcolemma in 5 of the 8 DCM hearts (62.5%);
these strong signals colocalized with both integrins. In all hearts,
CAR was detectable in subendothelial layers of the vessel wall, but
not on the luminal endothelial surface, and on interstitial cells. Human
CAR (hCAR) expressed in rat cardiomyocytes was targeted to cell-cell
contacts, which resembled CAR localization in DCM hearts and resulted
in 15-fold increased adenovirus uptake.

CONCLUSIONS: Low hCAR abundance may render normal human myocardium
resistant to CAR-dependent viruses, whereas re-expression of hCAR, such
as that observed in DCM, may be a key determinant of cardiac susceptibility
to viral infections. Asymmetric expression of hCAR in the vessel wall
may be an important determinant of adenovirus tropism in humans. hCAR
subcellular localization in human myocardium and hCAR targeting to cell-cell
contacts in cardiomyocyte cultures suggest that hCAR may play a role
in cell-cell contact formation.

Three cases of acute flaccid paralysis (AFP) associated with circulating
vaccine-derived poliovirus (cVDPV) isolates were reported in the Philippines
during March 15-July 26, 2001. The first case-patient, a child aged
8 years from northern Mindanao island (500 miles south of Manila) who
had received 3 doses of oral polio vaccine (OPV), had onset of paralysis
on March 15. A second child, aged 3 years from Laguna province on Luzon
island (60 miles south of Manila) who had received 3 OPV doses, presented
with signs of meningitis but no paralysis on July 23. A third child,
aged 14 months from Cavite province (25 miles from Manila and 45 miles
north of Laguna province) who had received 2 OPV doses, had onset of
paralysis on July 26. No patients had traveled outside of their province
of residence since birth. Characterization of isolates from the three
patients revealed type 1 polioviruses derived from Sabin vaccine strain
type 1, with a 3% genetic sequence difference between Sabin 1 vaccine
and vaccine-derived poliovirus (VDPV) isolates.

The three polioviruses are not identical but are closely related (>99%
sequence homology); they also appear to share an identical recombination
site with a nonpolio enterovirus in the noncapsid region of the genome.

BACKGROUND: Conventional approaches to virus detection failed to provide
convincing evidence of a viral etiology in sudden unexplained deaths
in infants (SUDI). Many viruses may not have been detected by the routinely
used methods; among them enteroviruses (EV) have seldom been found in
SUDI.

METHODS: In this study EV were sought directly in stools, in pharyngeal
and tracheal samples and in myocardial and lung tissues, by using a
nested PCR; they were also sought indirectly by detecting IgM antibodies
with a new capture immunoassay. Twenty-four SUDI cases were divided
into two groups: Group I, certainly associated with; or Group II, not
associated with clinical, biologic or histologic signs of viral infection.

RESULTS: EV were found in stools but their prevalence was not significantly
different between Group I and Group II (20 and 22.2%, respectively).
On the contrary EV were detected in respiratory tract and/or lung samples
in 53.8% of infants of Group I and in none of Group II. Anti-EV IgM
antibodies were detected in 55.5% of infants of Group I and in none
of Group II.

CONCLUSIONS: These results indicate that EV infection may be specifically
associated with the subgroup of SUDI with viral signs, raising the question
of its role in this condition.

PATHOGENESIS

CHARACTERIZATION

COXSACKIE VIRUS

Secondary heterotypic versus homotypic infection by
Coxsackie B group viruses: impact on early and late histopathological
lesions and virus genome prominence.

The impact of prior exposure to a different or identical strain of
Coxsackievirus B (CVB) on murine CVB myocarditis was studied using a
susceptible murine host (A/J[H-2a]) and myocarditic CVB3 or avirulent
CVB2 as primary or secondary infectants.

The effects of secondary heterotypic infection (CVB2 followed by CVB3)
and homotypic infection (CVB3 followed by CVB3) 28 days after primary
inoculation, versus CVB2 or CVB3 alone, on injury and viral genomic
replication, both early (day 7) and late (days 28 and 56), were evaluated.
After the primary infection by CVB2, trivial viral RNA was present in
the heart and other organs, and a substantial positivity was observed
with CVB3 infection. Seven days after secondary heterotypic (CVB2-CVB3)
infection, the quantity of CVB genome in heart, pancreas, liver, and
spleen was increased compared with the virus genome in the CVB3-CVB3
group and in the group with primary CVB3 infection alone. This phenomenon
was seen in the heart and spleen up to day 28 postsecondary infection.
Tissue inflammation and necrosis in heart and pancreas were prominent
7 days postsecondary infection with CVB2-CVB3 and correlated well with
an increased quantity of CVB genome. Virus genome was present in heart
and spleen 28 days after CVB3 infection alone. Serum CVB3 neutralization
titer was increased to 1:128 in CVB2-CVB3 group at days 7 and 28 postsecondary
infection, and serum completely neutralized cytopathological effects
of CVB3 in the CVB3-CVB3 group at day 7 and 28 postsecondary infection.

Our results indicate that secondary heterotypic infection by CVB causes
increased injury, inflammation, and CVB replication in target organs
such as the heart and pancreas, as well as in immune compartments like
the spleen. Compared with CVB3 alone, the intense inflammatory infiltriate
in the CVB2-CVB3 group is as not due solely to postviral sensitization
of the immune system, but rather to the inability of the host to eradicate
the virus.

Coxsackievirus infection of the pancreas: evaluation
of receptor expression, pathogenesis, and immunopathology.

Coxsackievirus type B (CVB) infection of the pancreas induces a massive
cellular infiltrate composed of natural killer cells, T cells, and macrophages
and leads to the destruction of exocrine tissue.

The physiological manifestations of pancreatic CVB infection are correlated
with viral tropism; the virus infects acinar cells but spares the islets
of Langerhans. Here we evaluate the mechanisms underlying pancreatic
inflammation and destruction and identify the determinants of viral
tropism. T-cell-mediated immunopathology has been invoked, along with
direct virus-mediated cytopathicity, to explain certain aspects of CVB-induced
pancreatic disease.

However, we show here that in the pancreas, the extent of inflammation
and tissue destruction appears unaltered in the absence of the cytolytic
protein perforin; these findings exclude any requirement for perforin-mediated
lysis by natural killer cells or cytotoxic T cells in CVB3-induced pancreatic
damage. Furthermore, perforin-mediated cytotoxic T-cell activity does
not contribute to the control of CVB infection in this organ. In addition,
we demonstrate that the recently identified coxsackie-adenovirus receptor
is expressed at high levels in acinar cells but is barely detectable
in islets, which is consistent with its being a major determinant of
virus tropism and, therefore, of disease. However, further studies using
various cell lines of pancreatic origin reveal secondary determinants
of virus tropism.

Enteroviruses may be involved in the pathogenesis of insulin-dependent
diabetes mellitus, either through direct beta-cell infection or as triggers
of autoimmunity.

In the present study we investigated the patterns of infection in adult
human islet cell preparations (consisting of 56+/-14% beta-cells) by
several coxsackieviruses. The cells were infected with prototype strains
of coxsackievirus B (CBV) 3, 4, and 5 as well as coxsackievirus A9 (CAV-9).
The previously characterized diabetogenic strain of coxsackievirus B4
(CBV-4-E2) was used as a reference. All viruses replicated well in beta-cells,
but only CBVs caused cell death. One week after infection, the insulin
response of the beta-cells to glucose or glucose plus theophylline was
most severely impaired by CBV-3 and CBV-5 infections. CBV-4 also caused
significant functional impairment, whereas CAV-9-infected cells responded
like uninfected controls. After 2 days of infection, about 40% of CBV-5-infected
cells had undergone morphological changes characteristic of pyknosis,
i.e. highly distorted nuclei with condensed but intact chromatin. Both
mitochondria and plasma membrane were intact in these cells. DNA fragmentation
was found in 5.9+/-1.1% of CBV-5-infected beta-cell nuclei (2.1+/-0.3%
in controls; P<0.01). CAV-9 infection did not induce DNA fragmentation.
One week after infection the majority of infected cells showed characteristics
of secondary necrosis. Medium nitrite and inducible nitric oxide synthase
messenger ribonucleic acid levels were not significantly up-regulated
by CBV infection. These results suggest that several enteroviruses may
infect human beta-cells. The infection may result in functional impairment
or death of the beta-cell or may have no apparent immediate adverse
effects, as shown here for CAV-9.

Coxsackie B viruses cause functional impairment and beta-cell death
characterized by nuclear pyknosis. Apoptosis appears to play a minor
role during a productive CBV infection in beta-cells.

A single-tube real-time reverse transcription-PCR (RT-PCR) assay for
enterovirus detection in cerebrospinal fluid (CSF) was developed based
on a fluorogenic probe and primers directed to highly conserved sequences
in the 5' untranslated region of the enterovirus genome.

Quantitative detection of enterovirus genome was demonstrated in a
linear range spanning at least 5 logs. Endpoint titration experiments
revealed that the in-tube detection limit of the assay was 11.8 enterovirus
genome equivalents (95% detection rate) corresponding in our current
extraction protocol to 592 enterovirus genome equivalents per ml of
CSF. Twenty CSF specimens not suspected of viral meningitis were all
found to be negative, and no cross-reactivity with herpes simplex virus
type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza
viruses A and B was observed. Nineteen CSF specimens from 70 patients
suspected of viral meningitis were determined to be positive by PCR
(27.1%), whereas only 17 were found to be positive by viral culture
(24.3%). The sensitivity of the assay was 100% and the specificity was
96.2% compared to viral culture. Data from the real-time RT-PCR assay
were available within 4 h.

Our data suggest that the novel real-time RT-PCR assay may offer a
reliable but significantly faster alternative to viral culture. Owing
to the elimination of postamplification detection steps, its conduct
required considerably less hands-on time and was associated with a substantially
reduced carryover risk compared to previously described PCR-based enterovirus
detection assays.

Comparison of a multiplex reverse transcription-pcr-enzyme
hybridization assay with conventional viral culture and immunofluorescence
techniques for the detection of seven viral respiratory pathogens.

One hundred forty-three respiratory specimens from 126 patients were
analyzed by RT-PCR-EHA, and the results were compared to those obtained
by conventional viral culture and immunofluorescence (IF) methods. RT-PCR-EHA
proved to be positive for 17 of 143 (11.9%) specimens, whereas 8 of
143 (5.6%) samples were positive by viral culture and/or IF. Eight samples
were positive by both RT-PCR-EHA and conventional methods, while nine
samples were RT-PCR-EHA positive and viral culture and IF negative.
Eight of the nine samples with discordant results were then independently
tested by a different multiplex RT-PCR assay for influenza virus types
A and B, and all eight proved to be positive.

In comparison to viral culture and IF methods, RT-PCR-EHA gave a sensitivity
and a specificity of 100 and 93%, respectively. Since RT-PCR-EHA was
able to detect more positive samples, which would otherwise have been
missed by routine methods, we suggest that this multiplex RT-PCR-EHA
provides a highly sensitive and specific means of diagnostic detection
of major respiratory viruses.

BACKGROUND: Enteroviruses are the most commonly identified cause of
viral meningitis. Detection of the enterovirus genome in cerebrospinal
fluid (CSF) using reverse-transcription polymerase chain reaction (PCR)
has proved to be useful in diagnosis and is more rapid and sensitive
than viral cultures. In routine practice, cytologic examination results
of CSF are obtained swiftly and PCR indication is performed as a second
step.

OBJECTIVES: The aim of this study was to determine, by analysis of
complete data from CSF results for 61 cases of proven enteroviral meningitis,
whether cytologic CSF findings can be used to establish viral etiology
and to indicate if PCR assay should be performed.

STUDY DESIGN: From a prospective study of children admitted during
1997 for suspected enterovirus meningitis in which PCR and viral cultures
of CSF were systematically performed, we selected 61 patients with proven
enterovirus meningitis. We compared global white cell count (WCC), relative
percentage of lymphocytes/neutrophils, PCR and culture for enterovirus,
patient age, and clinical data.

RESULTS: 92% of patients (56/61) had positive PCR in CSF and in 48%
(29/61) enterovirus was isolated in CSF. Nine patients (14.75%) had
WCC<10/mm(3); eight of them had positive PCR and two had positive culture.
There were comparable numbers of CSF with a predominance of lymphocytes
(n=25) and CSF with a predominance of neutrophils (n=22), and of positive
PCR and positive cultures of CSF in the two groups. Results were not
influenced by the age of the patients.

CONCLUSION: Irrespective of other CSF parameters, it seems difficult
to dispense with PCR assay for enterovirus genome detection. It should
be introduced as a true rapid routine test. Early reporting of a positive
PCR result could result in a considerable saving in health resources.

Enteroviruses usually cause self-limited disease that, although associated
with high morbidity, is rarely fatal. In certain patient populations,
however, the enteroviruses may cause potentially life-threatening infections.
Pleconaril is a novel compound that integrates into the capsid of picornaviruses,
including enteroviruses and rhinoviruses, preventing the virus from
attaching to cellular receptors and uncoating to release RNA into the
cell.

Pleconaril was used on a compassionate-release basis to treat patients
with potentially life-threatening enterovirus infections, and for 38
of these patients sufficient follow-up data were available for determining
responses to therapy. Response was evaluated in 4 categories: clinical,
virological, laboratory, and radiological. Most patients (28 [78%] of
36), including 12 of 16 with chronic enterovirus meningoencephalitis,
were judged to have a clinical response temporally associated with pleconaril
therapy.

Similarly, nearly all patients whose virological responses (12 [92%]
of 13), laboratory responses (14 [88%] of 16), and radiological responses
(3 [60%] of 5) could be evaluated were judged to have responded favorably
to a course of pleconaril treatment. Adverse effects were minimal and
the drug was generally well-tolerated.